Oligodeoxy nucleotide for preparing drugs for inhibiting tumor growth and application thereof

ABSTRACT

The present invention provides an oligodeoxy nucleotide for preparing drugs for inhibiting tumor growth and an application thereof. The core sequence of the oligodeoxy nucleotide is TTYSGGAAWT, wherein Y is C or T; S is C or G; and W is A or T. The oligodeoxy nucleotide above further comprises an antisense strand and a modified type thereof. The oligodeoxy nucleotide according to the present invention plays a role of inhibiting the tumor growth in vitro and vivo, and is expected to be used for preparing drugs for inhibiting tumor growth, with high specificity and high inhibition ratio.

TECHNICAL FIELD

The present invention belongs to the biological medicine field, andspecifically relates to an oligodeoxy nucleotide for preparing drugs forinhibiting tumor growth and an application thereof.

BACKGROUND

An oligodeoxy nucleotide may include an antisense nucleic acid, a smallinterfering RNA, an aptamer, a microRNA, a DECOY nucleic acid, or thelike, which respectively play biological functions thereof according todifferent effect target spots and mechanisms. The oligodeoxy nucleotideused as a therapeutic drug has been listed in USA. Meanwhile, dozens ofdrugs are under clinical research. With the progress of biotechnology,drug screening and preparation and other technologies, more and moreoligodeoxy nucleotides with specific functions are discovered, and aredeveloped and applied for treating, preventing and diagnosing variousdiseases.

BRIEF DESCRIPTION

Object of the invention: the present invention is intended to provide anoligodeoxy nucleotide for preparing drugs for inhibiting tumor growthand an application thereof. Through screening series oligodeoxynucleotides Oligo dsDNA, and testing the cancer inhibition activity theoligodeoxy nucleotides of the sequences 1-10 have an inhibiting effecton the tumor growth in vitro and vivo.

Technical solution: In order to achieve the technical object above, thepresent invention provides an oligodeoxy nucleotide for preparing drugsfor inhibiting tumor growth, wherein the oligodeoxy nucleotide is anyone of the following sequences 1-10:

sequence 1: 5′-CTTGAGGGGAATTTCCCAG-3′ sequence 2:5′-GAGAGGGGACTTTCCGAGAG-3′ sequence 3: 5′-CCTTGAAGGGATTTCCCTCC-3′Sequence 4: 5′-GCCATTTCCGGGAATTGCTA-3′ sequence 5: 5′-AGTTCTGGGAATTCC-3′sequence 6: 5′-AGTCATTTCCGGGAAATGACT-3′ sequence 7:5′-TGACTATTTCCCGCGACTT-3′ sequence 8: 5′-TTGACTATTTCCCGCCACTT-3′sequence 9: 5′-ATCTATTTCGCGCCCTTATG-3′ and sequence 10:5′-TTAAGTTTCGCGCCCTTTCTC-3′.

The oligodeoxy nucleotide may also an antisense strand of the sequences1-10 above.

As another embodiment, the oligodeoxy nucleotide is a modified type ofthe oligodeoxy nucleotide above.

To be specific, the modified type is special locus phosphorothioatemodification; or phosphorothioate modification with three nucleotides attwo ends; or full phosphorothioate modification.

Preferably, the modified locus corresponding to each sequence is asfollows:

sequence 1 (positive): 5′-CTTGAGGGGAATTTCCCAG-3′ sequence 2 (positive):5′-GAGAGGGGACTTTCCGAGAG-3′ sequence 3 (positive):5′-CCTTGAAGGGATTTCCCTCC-3′ sequence 4 (positive):5′-GCCATTTCCGGGAATTGCTA-3′ sequence 5 (positive): 5′-AGTTCTGGGAATTCC-3′sequence 6 (positive): 5′-AGTCATTTCCGGGAAATGACT-3′sequence 7 (positive): 5′-TGACTATTTCCCGCGACTT-3′ sequence 8 (positive):5′-TTGACTATTTCCCGCCACTT-3′ sequence 9 (positive):5′-ATCTATTTCGCGCCCTTATG-3′ and sequence 10 (positive):5′-TTAAGTTTCGCGCCCTTTCTC-3′

Wherein, the positive means that the positive-sense strand is listedonly, and the antisense strand is omitted.

The present invention proposes an application of the oligodeoxynucleotide used for preparing the drug for inhibiting tumor growth atthe same time.

To be specific, the following steps are comprised: purifying theoligodeoxy nucleotide through HPLC, and preparing the oligodeoxynucleotide into concentration of 1-2 mg/mL by sterilization water, thenconducting equal molar ratio mixture to the oligodeoxy nucleotide withan antisense strand thereof, heating the mixture to 80-85° C. for 10-15min through water bath, cooling the mixture to indoor temperaturenaturally, and annealing to combine the mixture into double strands forpreparing drugs for inhibiting tumor growth.

Wherein, the tumor is any one of lung cancer, breast cancer andpancreatic cancer.

Beneficial effects: the oligodeoxy nucleotide provided by the presentinvention plays a role of inhibiting the tumor growth in vitro and vivo,and is expected to be used for preparing drugs for inhibiting tumorgrowth, with high specificity and high inhibition ratio.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of a sequence 5 (KT17) to growth volumechange of human breast cancer cell MDA-MB-231 xenograft tumor in nudemice;

FIG. 2 is a schematic diagram of an inhibiting effect of the sequence 5(KT17) to the growth of human breast cancer cell MDA-MB-231 xenografttumor in nude mice;

FIG. 3 is a photo of the sequence 5 (KT17) to the growth volume changeof the human breast cancer cell MDA-MB-231 xenograft tumor in nude mice;

FIG. 4 is a schematic diagram of a sequence 8 (KT17) to growth volumechange of a human pancreatic cancer cell CFPAC-1 xenograft tumor in nudemice; and

FIG. 5 is a schematic diagram of an inhibiting effect of a sequence 1(KT17) to the growth of a human lung cancer cell NCI-H1975 xenografttumor in nude mice.

DETAILED DESCRIPTION

The oligodeoxy nucleotide provided by the present invention mainlyincludes three types which include 10 sequences in total, and arespecifically as follows:

First type: Oligo dsDNA mainly composed of a core sequence RGGGAHTTYCScomprises:

sequence 1 (positive): 5′-CTTGAGGGGAATTTCCCAG-3′ sequence 2 (positive):5′-GAGAGGGGACTTTCCGAGAG-3′ and sequence 3 (positive):5′-CCTTGAAGGGATTTCCCTCC-3′

Second type: Oligo dsDNA mainly composed of a core sequence TTYSGGAAWTcomprises:

sequence 4 (positive): 5′-GCCATTTCCGGGAATTGCTA-3′ sequence 5 (positive):5′-AGTTCTGGGAATTCC-3′ and sequence 6 (positive):5′-AGTCATTTCCGGGAAATGACT-3′

Third type: Oligo dsDNA mainly composed of a core sequence TTTCSCGCScomprises:

sequence 7 (positive): 5′-TGACTATTTCCCGCGACTT-3′ sequence 8 (positive):5′-TTGACTATTTCCCGCCACTT-3′ sequence 9 (positive):5′-ATCTATTTCGCGCCCTTATG-3′ and sequence 10 (positive):5′-TTAAGTTTCGCGCCCTTTCTC-3′.

Wherein, the positive means that the positive-sense strand is listedonly, and the antisense strand is omitted; in the modified types, thefirst type is part locus phosphorothioate modification, and theunderline portion is a modified locus, while the other type isphosphorothioate modification; or phosphorothioate modification withthree nucleotides at two ends; or full phosphorothioate modification,R=G/A; H=A/C/T; Y=C/T; S=C/G; and W=A/T.

The inhibiting effect of the oligodeoxy nucleotide above to tumors isillustrated hereinafter through specific embodiments.

Embodiment 1 Inhibiting effect of oligodeoxy nucleotide to tumor growthin vitro.

The oligodeoxy nucleotides of the combined sequences 1 to sequence andthe and the antisense strands thereof were subjected to three types ofdifferent structure modifications at the same time, which were threegroups including special locus phosphorothioate (as mentioned above),three base phosphorothioates at two ends, and full phosphorothioate.After the oligodeoxy nucleotides were purified through to purity morethan 90%, and prepared to concentration of 1 mg/mL by sterilizationwater; then equal molar ratio mixture was conducted to thepositive-sense and antisense strands, and the mixture was heated to80-85° C. for 10-15 min through water bath, cooled to indoor temperaturenaturally, and annealed to combine the mixture into double strands forstandby application.

Breast cancer cell strains MDA-MB-231, lung cancer cell strainsNCI-H1975, and pancreatic cancer cell strains CFPAC-1 were cultivatedrespectively in vitro according to a conventional method. When the cellswere grown to 10⁵-10⁶/mL the cells were dissociated and collected,counted and prepared into a cell suspension with concentration of3-5×10⁴/mL, and 100 μL cell suspension was added into each hole in a96-hole cell culture plate (3-5×10³ cells for each hole). The 96-holecell culture plate was put into a 5% CO₂ incubator under 37° C. for 24h; each group of the oligodeoxy nucleotides was diluted to theconcentration needed by complete medium, 100 μL corresponding mediumcontaining the oligodeoxy nucleotide was added in each hole, and anegative control group and a positive control group were set at the sametime, wherein the negative control group was normal saline, the positivecontrol group is paclitaxel, and a transfection method was subject to aproduct specification of Lipofectimine; the 96-hole cell culture platewas put into a 5% CO₂ under 37° C. for 72 h; 20 μL MTT (5 mg/mL) wasadded in each hole to cultivate for 4 h in the incubator; the culturemedium was discarded, 150 μL DMSO was added in each hole fordissolution, and was evenly and slightly mixed for 10 min throughshaking; then an OD value of each hole was read by a microplate reader(λ=490 nm) to calculate the inhibition ratio.

Method for calculating inhibition ratio of each group:

Inhibition ratio of experimental group=(OD value of negative controlgroup−OD value of experimental group/OD value of positive controlgroup)×100%

The result of screening in vitro showed that all the three types ofmodification groups of the sequences 1-10 had a certain inhibitingeffect to the tumor cell growth, wherein the special locusphosphorothioate modification group of the sequences 1-10 (see theunderlined parts in the sequence table for the phosphorothioate locus)had a better growth inhibiting effect to different tumor cells in vitro.The test result was as shown in Table 1:

TABLE 1 Half inhibition ratio IC₅₀ Unit: μM Sample Lung mammarypancreatic cancer cell cancer cell cancer cell strain strain strainNCI-H1975 MDA-MB-231 CFPAC-1 Sequence 1 3.663 1.492 7.982 Sequence 23.183 2.663 2.965 Sequence 3 4.062 5.831 3.225 Sequence 4 1.673 2.5731.672 Sequence 5 1.038 0.084 0.298 Sequence 6 1.329 1.588 1.661 Sequence7 1.699 3.438 2.306 Sequence 8 0.456 0.184 1.278 Sequence 9 5.497 3.0241.137 Sequence 10 4.219 3.31 1.442

The experimental results of partial secondary screening were as shown inTable 2 and Table 3:

TABLE 2 Mammary cancer cell strain MDA-MB-231 72 h Group Inhibitionratio Negative control 0 positive control 87.41% Sequence 5 200 nM82.55% IC50 = 100 nM 54.58% 77.342 nM 50 nM 31.35% 25 nM 17.29% 12.5 nM13.57% 6.25 nM 6.99% 3.125 nM 7.90% 1.5625 nM 1.91%

TABLE 3 Pancreatic cancer cell strain CFPAC-1 72 H Group Inhibitionratio Negative control positive control 88.41% Sequence 8 200 nM 62.73%IC50 = 100 nM 56.55% 94.577 nM 50 nM 41.86% 25 nM 15.47% 12.5 nM 1.59%6.25 nM 0.13% 3.125 nM −0.83% 1.5625 nM −1.64%

Embodiment 2: Inhibiting Effect of Oligodeoxy Nucleotide to Growth ofHuman Deviated Xenograft Tumor in Animal Body

The nucleic acid samples of the sequence 1, the sequence 5 and thesequence 8 (special locus modification groups) were respectivelyprepared into cationic liposome preparation, and five groups of controldrugs including a paclitaxel control group (positive control group) anda normal saline control group (negative control group), a low dosegroup, a medium dose group and a high dose group were selected. Testedanimals were female BALB/c nude mice (12-15 g, 4-5 weeks), etc. Thecultivated tumor cell suspension was collected and inoculated underskins at the right sides of axillae of the nude mice, then the animalswere grouped randomly when the tumor grown to 50-75 mm³, and each grouphad eight animals. Meanwhile, each group of the nude mice was dosed foronce each day for 14-18 days. A dosing method was tail vein injection,and the antineoplastic effects of the tested samples were dynamicallyobserved by a method of measuring tumor diameters. The nude mice wereput to death after being dosed for 14-18 days, and tumor blocks weretaken by operation for weighting and photographing.

A formula of calculating the tumor volume (TV) was as follows:

TV=½×a×b ²

Wherein, a and b respectively represented length and width.

The relative tumor volume (RTV) was calculated according to themeasurement results, and the calculation formula was as follows:

RTV=Vt/V0,

Wherein V0 was the tumor volume measured when dosing by cage (i.e., d0),and Vt was the tumor volume during each measurement.

The evaluation index of the antineoplastic activity: relative tumorincrease rate T/C (%), and the calculation formula was as follows:

T_(RTV)

T/C(%)=×100

C_(RTV)

T_(RTV): treatment group RTV; and C_(RTV): model group RTV

The average value was represented by X±SD, the statistical treatment wasconducted to the analysis between groups by t inspection, and SPSS(Staffstical Package for the Social Science) 17.0 was applied to conductstatistical analysis to the results.

The results of the pharmacodynamic experiment in vivo above showed thatthe inhibition ratios of a nucleic acid sequence 5 (KT17) to the high,medium and low dose groups of the breast cancer cells in vivorespectively reached to: 47.13%, 66.42% and 41.81%. As shown in FIGS.1-3, the inhibition ratios of a nucleic acid sequence 8 (KT59) to thehigh, medium and low dose groups of the breast cancer cell in vivorespectively reached to: 64.36%, 57.89% and 44.15%; the tumor inhibitionratio was relatively high and there was a certain dose-effectrelationship. As shown in FIG. 4, the inhibition ratios of a nucleicacid sequence 1 (KT32) to the high and low dose groups of the breastcancer cell in vivo respectively reached to: 56.35% and 49.44% (as shownin FIG. 5).

In conclusion, the sequences provided by the present invention haveobvious inhibiting effect on the tumor growth, and may be selected ascandidate drugs for further development.

1. An oligodeoxy nucleotide for preparing drugs for inhibiting tumorgrowth, wherein the oligodeoxy nucleotide is any one of the followingsequences 1-3: 5′-GCCATTTCCGGGAATTGCTA-3′ 5′-AGTTCTGGGAATTCC-3′5′-AGTCATTTCCGGGAAATGACT-3′


2. The oligodeoxy nucleotide according to claim 1, wherein theoligodeoxy nucleotide is an antisense strand of the three sequences inclaim
 1. 3. The oligodeoxy nucleotide according to claim 1, wherein theoligodeoxy nucleotide is a modified type of the oligodeoxy nucleotide inclaim
 1. 4. The oligodeoxy nucleotide according to claim 3, wherein themodified type is special locus phosphorothioate modification; orphosphorothioate modification with three nucleotides at two ends; orfull phosphorothioate modification.
 5. An application of the oligodeoxynucleotide according to claim 1 in preparing drugs for inhibiting tumorgrowth.
 6. The application according to claim 5, comprising thefollowing steps of purifying the oligodeoxy nucleotide through HPLC, andpreparing the oligodeoxy nucleotide into concentration of 1-2 mg/mL bysterilization water, then conducting equal molar ratio mixture to theoligodeoxy nucleotide with an antisense strand thereof, heating themixture to 80-85° C. for 10-15 min through water bath, cooling themixture to indoor temperature naturally, and annealing to combine themixture into double strands for preparing drugs for inhibiting tumorgrowth.
 7. The application according to claim 5, wherein the tumor isany one of lung cancer, breast cancer and pancreatic cancer.
 8. Theoligodeoxy nucleotide according to claim 2, wherein the oligodeoxynucleotide is a modified type of the oligodeoxy nucleotide in claim 2.9. The oligodeoxy nucleotide according to claim 8, wherein the modifiedtype is special locus phosphorothioate modification; or phosphorothioatemodification with three nucleotides at two ends; or fullphosphorothioate modification.
 10. An application of the oligodeoxynucleotide according to claim 2 in preparing drugs for inhibiting tumorgrowth.
 11. An application of the oligodeoxy nucleotide according toclaim 3 in preparing drugs for inhibiting tumor growth.
 12. Anapplication of the oligodeoxy nucleotide according to claim 4 inpreparing drugs for inhibiting tumor growth.